The purpose of this five-year proposal is to provide an integrative and personalized training program for the applicant to transition into an independent academic position as a physician-scientist in pediatric cardiology. The long-term goal is to discover novel loci associated with congenital heart malformations and to understand their function and interactions, in order to improve genetic testing, model risk, and predict outcomes for CHD in patient populations. The applicant has a strong background in genomics, human genetics, and next-generation sequencing studies with facility at the bench in all aspects of genetic analysis, the latest computational genetics tools, and software development capabilities. This career development plan (K99 phase) will provide additional training in cardiac developmental biology employing mouse models of heart formation, as well as sophisticated gene-targeting approaches to investigate the function of novel loci and gene networks in causing cardiac malformations. The applicant will also receive a wealth of informal and didactic training at Stanford University in specialized areas such as investigation of cardiac morphogenesis, and professional development, which will be critical for the applicant to gain autonomy and launch a productive career as an independent physician-scientist investigator. Under the expert mentorship of Dr. Thomas Quertermous, MD and Sean Wu, MD PhD along with the assembled advisory committee (Dr. Ashley, Dr. Rabinovitch, and Dr. Moskowitz) the applicant will receive the necessary guidance and resources to accomplish these goals and efficiently transition to independence during the R00 phase. The research topic of this proposal fulfills a significant knowledge gap in the field by identifying novel loci related to congenital hart disease and modeling their interactions in vivo to better understand the risk for disease in human populations. Congenital heart disease (CHD) is the most common congenital malformation and most disease is thought to be genetic in origin. Recent exome-sequencing studies have begun to identify new genes related to CHD, with a surprisingly low discovery rate of 10%. The applicant employed novel techniques to discover genetic variants in 30% of CHD patients and multiple variants within most affected individuals. Among these variants was a de novo mutation in a transcriptional repressor not previously linked to cardiac development. Preliminary knockout data of this gene shows heart defects in Drosophila and mouse, suggesting it has a conserved role in cardiac development. Deciphering the role of this novel gene in the complex process of heart development will be achieved by careful comparison of developing cardiac structures in the mouse knockout, along with RNA- sequencing studies to characterize the transcriptional networks altered by the absence of this highly conserved transcriptional repressor. The second component of the application is to discover novel CHD loci and model their interactions in an in vivo model system. Using 700 trios collected and genotyped by the PCGC now available to the general research community, highly sensitive informatics and powerful statistical tests will be applied to identify novel variants, loci, and gee networks related to CHD. To further investigate the interactions of multiple variants loci to cause disease, multiple genes comprising a network will be knocked out using a simultaneous CRISPR/Cas9 and the mice screened with for genotype and cardiac malformations delineate the impact of each gene individually and in combination. These experiments will illuminate the interactions between these loci and provide a model for the oligogenic basis of human cardiac malformations. Ultimately, this work will shed new light on novel genes and networks associated with risk for CHD, which is the first step in developing diagnostic and predictive genetic testing for patient care.